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  Apparatus and method for sequencing a nucleic acidUSPTO Application #: 20070092872Title: Apparatus and method for sequencing a nucleic acid Abstract: Disclosed herein are methods and apparatuses for sequencing a nucleic acid. These methods permit a very large number of independent sequencing reactions to be arrayed in parallel, permitting simultaneous sequencing of a very large number (>10,000) of different oligonucleotides. (end of abstract) Agent: Mintz Levin Cohn Ferris Glovsky & Popeo - New York, NY, US Inventors: Jonathan M. Rothberg, Joel S. Bader, Scott B. Dewell, Keith McDade, John W. Simpson, Jan Berka, Christopher M. Colangelo, Michael P. Weiner USPTO Applicaton #: 20070092872 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070092872. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] RELATED APPLICATIONS [0002] This application claims the benefit of priority to U.S. Ser. No. 10/104,280 filed Mar. 21, 2002; which is a CIP of 09/814,338 filed Mar. 21, 2001; which is a CIP of U.S. Ser. No. 09/664,197 filed Sep. 18, 2000; which is a CIP of U.S. Ser. No. 09/398,833 filed Sep. 16, 1999, now U.S. Pat. No. 6,274,320. Each of the above referenced patent and patent applications are incorporated herein by reference in their entireties. FIELD OF THE INVENTION [0003] The invention relates to apparatus and methods for determining the sequence of a nucleic acid. BACKGROUND OF THE INVENTION [0004] Many diseases are associated with particular DNA sequences. The DNA sequences are often referred to as DNA sequence polymorphisms to indicate that the DNA sequence associated with a diseased state differs from the corresponding DNA sequence in non-afflicted individuals. DNA sequence polymorphisms can include, e.g., insertions, deletions, or substitutions of nucleotides in one sequence relative to a second sequence. An example of a particular DNA sequence polymorphism is 5'-ATCG-3, relative to the sequence 5'-ATGG-3' at a particular location in the human genome. The first nucleotide `G` in the latter sequence has been replaced by the nucleotide `C` in the former sequence. The former sequence is associated with a particular disease state, whereas the latter sequence is found in individuals not suffering from the disease. Thus, the presence of the nucleotide sequence `5-ATCG-3` indicates the individual has the particular disease. This particular type of sequence polymorphism is known as a single-nucleotide polymorphism, or SNP, because the sequence difference is due to a change in one nucleotide. [0005] Techniques which enable the rapid detection of as little as a single DNA base change are therefore important methodologies for use in genetic analysis. Because the size of the human genome is large, on the order of 3 billion base pairs, techniques for identifying polymorphisms must be sensitive enough to specifically identify the sequence containing the polymorphism in a potentially large population of nucleic acids. [0006] Typically a DNA sequence polymorphism analysis is performed by isolating DNA from an individual, manipulating the isolated DNA, e.g., by digesting the DNA with restriction enzymes and/or amplifying a subset of sequences in the isolated DNA. The manipulated DNA is then examined further to determine if a particular sequence is present. [0007] Commonly used procedures for analyzing the DNA include electrophoresis. Common applications of electrophoresis include agarose or polyacrylamide gel electrophoresis. DNA sequences are inserted, or loaded, on the gels and subjected to an electric field. Because DNA carries a uniform negative charge, DNA will migrate through the gel based on properties including sequence length, three-dimensional conformation and interactions with the gel matrix upon application of the electrical field. In most applications, smaller DNA molecules will migrate more rapidly through the gel than larger fragments. After electrophoresis has been continued for a sufficient length of time, the DNA molecules in the initial population of DNA sequences will have been separated according to their relative sizes. [0008] Particular DNA molecules can then be detected using a variety of detection methodologies. For some applications, particular DNA sequences are identified by the presence of detectable tags, such as radioactive labels, attached to specific DNA molecules. [0009] Electrophoretic-based separation analyses can be less desirable for applications in which it is desirable to rapidly, economically, and accurately analyze a large number of nucleic acid samples for particular sequence polymorphisms. For example, electrophoretic-based analysis can require a large amount of input DNA. In addition, processing the large number of samples required for electrophoretic-based nucleic acid based analyses can be labor intensive. Furthermore, these techniques can require samples of identical DNA molecules, which must be created prior to electrophoresis at costs that can be considerable. [0010] Recently, automated electrophoresis systems have become available. However, electrophoresis can be ill suited for applications such as clinical sequencing, where relatively cost-effective units with high throughput are needed. Thus, the need for non-electrophoretic methods for sequencing is great. For many applications, electrophoresis is used in conjunction with DNA sequence analysis. [0011] Several alternatives to electrophoretic-based sequencing have been described. These include scanning tunnel electron microscopy, sequencing by hybridization, and single molecule detection methods. [0012] Another alternative to electrophoretic-based separation analysis is solid substrate-based nucleic acid analyses. These methods typically rely upon the use of large numbers of nucleic acid probes affixed to different locations on a solid support. These solid supports can include, e.g., glass surfaces, plastic microtiter plates, plastic sheets, thin polymers, or semi-conductors. The probes can be, e.g., adsorbed or covalently attached to the support, or can be microencapsulated or otherwise entrapped within a substrate matrix, membrane, or film. [0013] Substrate-based nucleic acid analyses can include applying a sample nucleic acid known or suspected of containing a particular sequence polymorphism to an array of probes attached to the solid substrate. The nucleic acids in the population are allowed to hybridize to complementary sequences attached to the substrate, if present. Hybridizing nucleic acid sequences are then detected in a detection step. [0014] Solid support matrix-based hybridization and sequencing methodologies can require a high sample-DNA concentration and can be hampered by the relatively slow hybridization kinetics of nucleic acid samples with immobilized oligonucleotide probes. Often, only a small amount of template DNA is available, and it can be desirable to have high concentrations of the target nucleic acid sequence. Thus, substrate based detection analyses often include a step in which copies of the target nucleic acid, or a subset of sequences in the target nucleic acid, is amplified. Methods based on the Polymerase Chain Reaction (PCR), e.g., can increase a small number of probe targets by several orders of magnitude in solution. However, PCR can be difficult to incorporate into a solid-phase approach because the amplified DNA is not immobilized onto the surface of the solid support matrix. [0015] Solid-phase based detection of sequence polymorphisms has been described. An example is a "mini-sequencing" protocol based upon a solid phase principle described by Hultman, et al., 1988. Nucl. Acid. Res. 17: 4937-4946; Syvanen, et al., 1990. Genomics 8: 684-692. In this study, the incorporation of a radiolabeled nucleotide was measured and used for analysis of a three-allelic polymorphism of the human apolipoprotein E gene. However, such radioactive methods are not well suited for routine clinical applications, and hence the development of a simple, highly sensitive non-radioactive method for rapid DNA sequence analysis has also been of great interest. SUMMARY OF THE INVENTION [0016] The invention is based in part on the use of arrays for determining the sequences of nucleic acids. [0017] Accordingly, in one aspect, the invention involves an array including a planar surface with a plurality of reaction chambers disposed thereon, wherein the reaction chambers have a center to center spacing of between 5 to 200 .mu.m and each chamber has a width in at least one dimension of between 0.3 .mu.m and 100 .mu.m. In some embodiments, the array is a planar surface with a plurality of cavities thereon, where each cavity forms an analyte reaction chamber. In a preferred embodiment, the array is fashioned from a sliced fiber optic bundle (i.e., a bundle of fused fiber optic cables) and the reaction chambers are formed by etching one surface of the fiber optic reactor array ("FORA"). The cavities can also be formed in the substrate via etching, molding or micromachining. [0018] Specifically, each reaction chamber in the array typically has a width in at least one dimension of between 0.3 .mu.m and 100 .mu.m, preferably between 0.3 .mu.m and 20 .mu.m, mst preferably between 0.3 .mu.m and 10 .mu.m. In a separate embodiment, we contemplate larger reaction chambers, preferably having a width in at least one dimension of between 20 .mu.m and 70 .mu.m. [0019] The array typically contains more than 1,000 reaction chambers, preferably more than 400,000, more preferably between 400,000 and 20,000,000, and most preferably between 1,000,000 and 16,000,000 cavities or reaction chambers. The shape of each cavity is frequently substantially hexagonal, but the cavities can also be cylindrical. In some embodiments, each cavity has a smooth wall surface, however, we contemplate that each cavity may also have at least one irregular wall surface. The bottom of each of the cavities can be planar or concave. [0020] The array is typically constructed to have cavities or reaction chambers with a center-to-center spacing between 10 to 150 .mu.m, preferably between 50 to 100 .mu.m. [0021] Each cavity or reaction chamber typically has a depth of between 10 .mu.m and 100 .mu.m; alternatively, the depth is between 0.25 and 5 times the size of the width of the cavity, preferably between 0.3 and 1 times the size of the width of the cavity. Continue reading... Full patent description for Apparatus and method for sequencing a nucleic acid Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Apparatus and method for sequencing a nucleic acid patent application. Patent Applications in related categories: 20080108057 - Allelic imbalance in the diagnosis and prognosis of cancer - Methods for assessing the extent of allelic imbalance in a genomic nucleic acid sample. 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